Design and synthesis of a sulphur containing Schiff base drug: DNA binding studies and theoretical calculations.

The Schiff base compound MTA ((E)-5-methyl-N'-((5-methylthiophen-2-yl)methylene)-1H-pyrazole-3-carbohydrazide) derived from 2-thiophenecarboxaldehyde and 5-methylpyrazole-3-carbohydrazide has been designed to develop new sulphur containing DNA targeted molecule. The MTA has been characterized by elemental analyses, 1H-NMR, single crystal X-ray diffraction studies as well as by geometry optimization of using DFT/B3LYP. The interaction of MTA with Calf thymus DNA (CT-DNA) was studied by spectroscopic and calorimetric techniques. The synthesized compound was found to bind with CT-DNA through groove binding mode, and the binding constant was estimated to be (4.15 ± 0.08) × 104 M-1. The negative ΔG0 and positive ΔS0 values obtained from the calorimetric technique confirmed the spontaneity of the binding of MTA with DNA.Communicated by Ramaswamy H. Sarma.

Interaction of the putative anticancer alkaloid chelerythrine with nucleic acids: biophysical perspectives.

Alkaloids represent an important group of molecules that have immense pharmacological potential. Benzophenanthridine alkaloids are one such class of alkaloids known for their myriad pharmacological activities that include potential anticancer activities. Chelerythrine is a premier member of the benzophenanthridine family of the isoquinoline group. This alkaloid is endowed with excellent medicinal properties and exhibits antibacterial, antimicrobial and anti-inflammatory properties. The molecular basis of its therapeutic activity is considered due to its nucleic acid binding capabilities. This review focuses on consolidating the current status on the nucleic acid binding properties of chelerythrine that is essential for the rational design and development of this alkaloid as a potential drug. This work reviews the interaction of chelerythrine with different natural and synthetic nucleic acids like double- and single-stranded DNAs, heat-denatured DNA, quadruplex DNA, double- and single-stranded RNA, tRNA and triplex and quadruplex RNA. The review emphasizes on the mode, specificity, conformational aspects and energetics of the binding that is particularly helpful for developing nucleic acid targeted therapeutics. The fundamental results discussed in this review will greatly benefit drug development for many diseases and serve as a database for the design of futuristic benzophenanthridine-based therapeutics.

Correction to "Pyridine Derivative of the Natural Alkaloid Berberine as Human Telomeric G4-DNA Binder: A Solution and Solid-State Study".

[This corrects the article DOI: 10.1021/acsmedchemlett.9b00516.].

Pyridine Derivative of the Natural Alkaloid Berberine as Human Telomeric G4-DNA Binder: A Solution and Solid-State Study.

Telomerase is an enzyme deputed to the maintenance of eukaryotic chromosomes; however, its overexpression is a recognized hallmark of many cancer forms. A viable route for the inhibition of telomerase in malignant cells is the stabilization of G-quadruplex structures (G4) at the 3' overhang of telomeres. Berberine has shown in this regard valuable G4 binding properties together with a significant anticancer activity and telomerase inhibition effects. Here, we focused on a berberine derivative featuring a pyridine containing side group at the 13th position. Such modification actually improves the binding toward telomeric G-quadruplexes and establishes a degree of selectivity in the interaction with different sequences. Moreover, the X-ray crystal structure obtained for the complex formed by the ligand and a bimolecular human telomeric quadruplex affords a better understanding of the 13-berberine derivatives behavior with telomeric G4 and allows to draw useful insights for the future design of derivatives with remarkable anticancer properties.

Polyamines and its analogue modulates amyloid fibrillation in lysozyme: A comparative investigation.

BACKGROUND: Polyamines can induce protein aggregation that can be related to the physiology of the cellular function. Polyamines have been implicated in protein aggregation which may lead to neuropathic and non neuropathic amyloidosis. SCOPE OF REVIEW: Change in the level of polyamine concentration has been associated with ageing and neurodegeneration such as Parkinson's disease, Alzheimer's disease. Lysozyme aggregation in the presence of polyamines leads to non neuropathic amyloidosis. Polyamine analogues can suppress or inhibit protein aggregation suggesting their efficacy against amyloidogenic protein aggregates. MAJOR CONCLUSIONS: In this study we report the comparative interactions of lysozyme with the polyamine analogue, 1-naphthyl acetyl spermine in comparison with the biogenic polyamines through spectroscopy, calorimetry, imaging and docking techniques. The findings revealed that the affinity of binding varied as spermidine > 1-naphthyl acetyl spermine > spermine. The biogenic polyamines accelerated the rate of fibrillation significantly, whereas the analogue inhibited the rate of fibrillation to a considerable extent. The polyamines bind near the catalytic diad residues viz. Glu35 and Asp52, and in close proximity of Trp62 residue. However, the analogue showed dual nature of interaction where its alkyl amine region bind in same way as the biogenic polyamines bind to the catalytic site, while the naphthyl group makes hydrophobic contacts with Trp62 and Trp63, thereby suggesting its direct influence on fibrillation. GENERAL SIGNIFICANCE: This study, thus, potentiates, the development of a polyamine analogue that can perform as an effective inhibitor targeted towards aggregation of amyloidogenic proteins.

Insights on the interaction of phenothiazinium dyes methylene blue and new methylene blue with synthetic duplex RNAs through spectroscopy and modeling.

The ubiquitous influence of double stranded RNAs in biological events makes them imperative to gather data based on specific binding procedure of small molecules to various RNA conformations. Particular interest may be attributed to situations wherein small molecules target RNAs altering their structures and causing functional modifications. The main focus of this study is to delve into the interactive pattern of two small molecule phenothiazinium dyes, methylene blue and new methylene blue, with three duplex RNA polynucleotides-poly(A).poly(U), poly(C).poly(G) and poly(I).poly(C) by spectroscopic and molecular modeling techniques. Analysis of data as per Scatchard and Benesi-Hildebrand methodologies revealed highest affinity of these dyes to poly(A).poly(U) and least to poly(I).poly(C). In addition to fluorescence quenching, viscometric studies also substantiated that the dyes follow different modes of binding to different RNA polynucleotides. Distortion in the RNA structures with induced optical activity in the otherwise optically inactive dye molecules was evidenced from circular dichroism results. Dye-induced RNA structural modification occurred from extended conformation to compact particles visualized by atomic force microscopy. Molecular docking results revealed different binding patterns of the dye molecules within the RNA duplexes. The novelty of the present work lies towards a new contribution of the phenothiazinium dyes in dysfunctioning double stranded RNAs, advancing our knowledge to their potential use as RNA targeted small molecules.

Nucleic acids binding strategies of small molecules: Lessons from alkaloids.

BACKGROUND: Nucleic acids are now important targets for therapeutic intervention. Alkaloids are an important class of molecules that have myriad therapeutic utility. Isoquinoline and benzophenanthridine alkaloids exhibit multiple pharmacological activities which are often related to their strong nucleic acid binding abilities. Therefore, a review of their interaction aspects with varying nucleic acid structures is essential for rational design and development as therapeutic agents. SCOPE OF THE REVIEW: This work reviews the interaction of various therapeutically important isoquinoline and benzophenanthridine alkaloids with nucleic acids. The review lends insights into the molecular aspects of the interaction that is critical from the perspective of designing better therapeutics. MAJOR CONCLUSIONS: This review provides a concise report on the recent developments and advancements on the interaction of various alkaloids with natural and synthetic nucleic acids. The review focuses on the mode, mechanism, specificity, conformational aspects and energetics of the interaction that will be helpful in the design and synthesis nucleic acid targeted alkaloid analogs. GENERAL SIGNIFICANCE: The molecular aspects of the interaction presented here will benefit the development of effective drugs for many diseases. The fundamental results discussed in this review can serve as a database for the design and development of futuristic nucleic acid based small molecule therapeutics.

Natural Aristolochia Alkaloid Aristololactam-ß-D-glucoside: Interaction with Biomacromolecules and Correlation to the Biological Perspectives.

BACKGROUND: Natural aristolochia alkaloids have attracted the attention of both chemists and biologists from the stand point of their structural and pharmacological aspects. Many of the compounds isolated in this group are potent tumor inhibitors. These are divided into nitrophenanthrinic acid, phenanthrene lactams and isoquinoline alkaloids. A number of structure-activity studies have been performed on aristolochia alkaloids. Of particular interest is the molecule with the ß-D-glucoside moiety that has similarity to the clinical anticancer agent daunomycin. OBJECTIVE: The anticancer activity of aristololactam-ß-D-glucoside has been thought to be due to its DNA and RNA binding activities among other actions. In this article we focus on the physicochemical property of this alkaloid and the structural and functional aspects of its binding to different nucleic acid and protein structures. METHODS: This review highlights a large number of biophysical studies employing various analytical techniques like absorbance, fluorescence, circular dichroism, thermal melting, viscosity, IR study, isothermal calorimetry and differential scanning calorimetry. RESULT: The detailed binding mechanism in terms of the structural and thermodynamic aspects at the molecular level has been discussed. CONCLUSION: This review enables to assess the high potential of developing aristololactam-ß-Dglucoside and related alkaloids as therapeutic agents.

Thionine Conjugated Gold Nanoparticles Trigger Apoptotic Activity Toward HepG2 Cancer Cell Line.

Cancer cells were locally damaged using targeted gold nanoparticles (GNP) conjugated with therapeutic dye thionine (TN). GNP was prepared by citrate reduction method, and the two complexes, namely GTN1 and GTN2, were synthesized by mixing GNP and TN at different ratios at room temperature and at 80 °C, respectively. It is expected that GTN1 is formed when stabilizer TN participates in the reduction of Au3+ ions to Au0 nanocrystallites, while GTN2 is synthesized when the cationic dye TN adsorbs onto the GNP surfaces due to the electrostatic attraction. The compounds were characterized by strong plasmon resonance absorption, Fourier transform infrared spectroscopy, dynamic light scattering technique, ζ-potential measurement, transmission electron microscopy, and atomic force microscopy. Crystallinity of the NPs was ascertained by X-ray diffraction. Strong binding of GTN1 to DNA and the structural perturbation prompted us to study the cytotoxic activity of the compounds on hepatocellular carcinoma cell lines (HepG2) by MTT assay. The mode of cytotoxicity was found due to reactive oxygen species (ROS) generation inside the cells. Fluorescence microscopy analysis revealed nuclear fragmentation which was caused due to the ROS. The GTN1 induced fragmentation led to the apoptosis mediated cell death as found from the cell cycle study. Conclusions drawn from these studies emphasized GTN1 to be capable of inhibiting proliferation in cancer cells in an amount greater than that of other compounds. The importance of the work lies in the exploration of effectiveness of nanoparticles to prevent cancer cell proliferation, which is a progressive step toward novel biomedical applications.

A new insight into the interaction of ZnO with calf thymus DNA through surface defects.

Experimental evidences on the binding interaction of ZnO and Calf Thymus (CT) DNA using several biophysical techniques are the centre of interest of the present study. The interaction of ZnO with CT DNA has been investigated in detail by absorption spectral study, fluorescence titration, Raman analysis, zeta potential measurement, viscometric experiment along with thermal melting study and microscopic analysis. Steady-state fluorescence study revealed the quenching (48%) of the surface defect related peak intensity of ZnO on interaction with DNA. The optimized concentration of ZnO and DNA to obtain this level of quenching has been found to be 0.049mM and 1.027µM, respectively. Additional fluorescence study with 8-hydroxy-5-quinoline (HQ) as a fluorescence probe for Zn2+ ruled out the dissolution effect of ZnO under the experimental conditions. DNA conjugation on the surface of ZnO was also supported by Raman study. The quantitative variation in conductivity as well as electrophoretic mobility indicated significant interaction of ZnO with the DNA molecule. Circular dichroism (CD) and viscometry titrations provided clear evidence in support of the conformational retention of the DNA on interaction with ZnO. The binding interaction was found to be predominantly entropy driven in nature. The bio-physical studies presented in this paper exploring ZnO-CT DNA interaction could add a new horizon to understand the interaction between metal oxide and DNA.

Influence of the ionic liquid 1-butyl-3-methylimidazolium bromide on amyloid fibrillogenesis in lysozyme: Evidence from photophysical and imaging studies.

Many proteins can abnormally fold to form pathological amyloid deposits/aggregates that are responsible for various degenerative disorders called amyloidosis. Here we have examined the anti-amyloidogenic potency of an ionic liquid, 1-butyl-3-methylimidazolium bromide, using lysozyme as a model system. Thioflavin T fluorescence assay demonstrated that the ionic liquid suppressed the formation of lysozyme fibrils significantly. This observation was further confirmed by the Congo red assay. Fluorescence microscopy, intrinsic fluorescence studies, nile red fluorescence assay, ANS binding assay and circular dichroism studies also testified diminishing of the fibrillogenesis in the presence of ionic liquid. Formation of amyloid fibrils was also characterized by α to ß conformational transition. From far-UV circular dichroism studies it was observed that the ß-sheet content of the lysozyme samples decreased in the presence of the ionic liquid which in turn implied that fibrillogenesis was supressed by the ionic liquid. Atomic force microscopy imaging unequivocally established that the ionic liquid attenuated fibrillogenesis in lysozyme. These results may be useful for the development of more effective therapeutics for amyloidosis.

Lipid chain saturation and the cholesterol in the phospholipid membrane affect the spectroscopic properties of lipophilic dye nile red.

We have studied the effect of composition and the phase state of phospholipid membranes on the emission spectrum, anisotropy and lifetime of a lipophilic fluorescence probe nile red. Fluorescence spectrum of nile red in membranes containing cholesterol has also been investigated in order to get insights into the influence of cholesterol on the phospholipid membranes. Maximum emission wavelength (λem) of nile red in the fluid phase of saturated and unsaturated phospholipids was found to differ by ~10nm. The λem was also found to be independent of chain length and charge of the membrane. However, the λem is strongly dependent on the temperature in the gel phase. The λem and rotational diffusion rate decrease, whereas the anisotropy and lifetime increase markedly with increasing cholesterol concentration for saturated phosoholipids, such as, dimyristoyl phosphatidylcholine (DMPC) in the liquid ordered phase. However, these spectroscopic properties do not alter significantly in case of unsaturated phospholipids, such as, dioleoyl phosphatidylcholine (DOPC) in liquid disordered phase. Interestingly, red edge excitation shift (REES) in the presence of lipid-cholesterol membranes is the direct consequences of change in rotational diffusion due to motional restriction of lipids in the presence of cholesterol. This study provides correlations between the membrane compositions and fluorescence spectral features which can be utilized in a wide range of biophysical fields as well the cell biology.

Biophysical studies on the interaction of a novel oxime based palladium(II) complex with DNA and RNA.

A novel oxime based palladium(II) complex (1) has been synthesized out of the reaction of a Schiff base ligand, 1-phenyl-1-(pyridin-2-yl-hydrazono)-propan-2-one oxime (LH2) with Na2[PdCl4] in THF. Red and diamagnetic 1 has thoroughly been characterized by several analytical and spectroscopic means like CHN, ESI-MS, FAB-MS, FT-IR, 1H NMR, 13C NMR, UV-Vis and molar electrical conductivity measurements. Geometry optimizations at the level of DFT reveals that the Pd(II) centre in 1 is nested in a square-planar 'N3Cl' coordination environment. Semi-empirical BVS mode of analysis was also undertaken to reproduce the oxidation state of the palladium centre. 1 shows quasi-reversible Pd(II)/Pd(III) and Pd(III)/Pd(IV) redox couples in its CV in acetonitrile. The photophysical studies reveal that 1 is two-fold less emissive than its tethering ligand. Several biophysical studies have been undertaken to demonstrate the binding aspects of DNA and RNA with 1. Ethidium bromide displacement study concludes that 1 is partially intercalated to the CT DNA. Thermodynamic parameters of binding have also been determined from temperature dependent fluorescence spectroscopy employing the van't Hoff plot. The binding constants as determined from McGhee-von Hippel equation (Scatchard plot) indicate that 1 is a good binder of both DNA and RNA. However, the magnitude of the binding constant as determined for RNA interaction with 1 is found to be higher than that for DNA interaction.

Exploring the interaction of phenothiazinium dyes methylene blue, new methylene blue, azure A and azure B with tRNAPhe: spectroscopic, thermodynamic, voltammetric and molecular modeling approach.

This study focuses on the understanding of the interaction of phenothiazinium dyes methylene blue (MB), new methylene blue (NMB), azure A (AZA) and azure B (AZB) with tRNAPhe with particular emphasis on deciphering the mode and energetics of the binding. Strong intercalative binding to tRNAPhe was observed for MB, NMB and AZB, bound by a partial intercalative mode. AZA has shown groove binding characteristics. From spectroscopic studies binding affinity values of the order of 105 M-1 were deduced for these dyes; the trend varied as MB > NMB > AZB > AZA. The binding was characterized by an increase of thermal melting temperatures and perturbation in the circular dichroism spectrum of tRNA. All the dyes acquired optical activity upon binding to tRNA. The binding was predominantly entropy driven with a favorable enthalpy term that increased with temperature in all the cases. Dissection of the Gibbs energy to polyelectrolytic and non-polyelectrolytic terms revealed a major role of the non-electrostatic forces in the binding. The small but significant heat capacity changes and the observed enthalpy-entropy compensation phenomenon confirmed the involvement of multiple weak non-covalent forces driving the interaction. The mode of binding was confirmed from quenching, viscosity and cyclic voltammetric results. Using density functional theory, ground state optimized structures of the dyes were calculated to provide insight into theoretical docking studies to correlate the experimental approaches. The modeling results verified the binding location as well as the binding energy of complexation. The results may provide new insights into the structure-activity relationship useful in the design of effective RNA targeted therapeutic agents.

Binding interaction of phenothiazinium dyes with double stranded RNAs: Spectroscopic and calorimetric investigation.

RNA targeting through small molecules is an emerging and promising therapeutic route that necessitates identification of small molecules that can selectively target specific RNA structures. In this context a comparative study of the interaction of two phenothiazinium dyes thionine (THN) and toluidine blue O (TBO) with three double stranded RNA polynucleotides (ds RNAs) viz. poly(I).poly(C), poly(A).poly(U) and poly(C).poly(G) was conducted by various biophysical techniques. A higher binding of THN with poly(I).poly(C) over poly(A).poly(U) and poly(C).poly(G) was observed. The intercalative binding and RNA induced fluorescence quenching of the dyes through a static mechanism was confirmed by viscosity studies and steady state polarization anisotropy experiments. Binding induced structural perturbation in the RNA polynucleotides was confirmed from circular dichroism spectroscopy. DSC and thermal melting experiments confirmed that the binding resulted in strong thermal stabilization. The binding affinity of THN with poly(I).poly(C) was the highest followed by that to poly(A).poly(U) and poly(C).poly(G). The trend was the same for TBO also, but THN bound stronger than TBO. The binding of the dyes was characterized by strong negative enthalpy changes with minimum positive entropy changes indicating typical intercalative interaction. The results presented here may be useful to design new types of RNA binding antitumor, antibacterial and anticancer agents.

A biophysical investigation on the binding of proflavine with human hemoglobin: Insights from spectroscopy, thermodynamics and AFM studies.

Interaction of proflavine with hemoglobin (Hgb) was studied employing spectroscopy, calorimetry, and atomic force microscopy. The equilibrium constant was found to be of the order 104M-1. The quenching of Hgb fluorescence by proflavine was due to the complex formation. Calculation of the molecular distance (r) between the donor (ß-Trp37 of Hgb) and acceptor (proflavine) suggested that energy can be efficiently transferred from the ß-Trp37 residue at the α1ß2 interface of the protein to the dye. Proflavine induced significant secondary structural changes in Hgb. Synchronous fluorescence studies showed that proflavine altered the microenvironment around the tryptophan residues to a greater extent than the tyrosine residues. Circular dichroism spectral studies showed that proflavine caused significant reduction in the α-helical content of Hgb. The esterase activity assay further complemented the circular dichroism data. The Soret band intensity of Hgb decreased upon complexation. Differential scanning calorimetry and circular dichroism melting results revealed that proflavine induced destabilization of Hgb. The binding was driven by both positive entropy and negative enthalpy. Atomic force microscopy studies revealed that the essential morphological features of hemoglobin were retained in the presence of proflavine. Overall, insights on the photophysical aspects and energetics of the binding of proflavine with Hgb are presented.

Sanguinarine and Its Role in Chronic Diseases.

The use of natural products derived from plants as medicines precedes even the recorded human history. In the past few years there were renewed interests in developing natural compounds and understanding their target specificity for drug development for many devastating human diseases. This has been possible due to remarkable advancements in the development of sensitive chemistry and biology tools. Sanguinarine is a benzophenanthridine alkaloid derived from rhizomes of the plant species Sanguinaria canadensis. The alkaloid can exist in the cationic iminium and neutral alkanolamine forms. Sanguinarine is an excellent DNA and RNA intercalator where only the iminium ion binds. Both forms of the alkaloid, however, shows binding to functional proteins like serum albumins, lysozyme and hemoglobin. The molecule is endowed with remarkable biological activities and large number of studies on its various activities has been published potentiating its development as a therapeutic agent particularly for chronic human diseases like cancer, asthma, etc. In this article, we review the properties of this natural alkaloid, and its diverse medicinal applications in relation to how it modulates cell death signaling pathways and induce apoptosis through different ways, its utility as a therapeutic agent for chronic diseases and its biological effects in animal and human models. These data may be useful to understand the therapeutic potential of this important and highly abundant alkaloid that may aid in the development of sanguinarine-based therapeutic agents with high efficacy and specificity.

Probing the binding of anticancer drug topotecan with human hemoglobin: Structural and thermodynamic studies.

Protein - ligand interactions play pivotal role in almost all the biological processes occurring in living organisms, and therefore such studies hold immense importance from the standpoint of rational drug design and development. In this study the binding of the topoisomerase I inhibitor drug, topotecan to hemoglobin was probed using various biophysical and microcalorimetry techniques. Spectrofluorimetric data confirmed the static nature of the quenching mechanism of the protein induced by the drug. Significant conformational changes in the protein were ascertained from circular dichroism and three dimensional fluorescence results. Synchronous fluorescence study revealed an increase in the polarity around the Trp residues of the protein while atomic force microscopy study enabled to obtain images of the bound molecules. Isothermal titration calorimetry studies indicated an exothermic binding with a negative Gibbs energy change; ionic strength variation suggested a greater contribution from non-polyelectrolytic forces in the binding process. Differential scanning calorimetry studies indicated an increased thermal stabilization of the protein upon topotecan binding which is also in close agreement with the results obtained from absorbance and circular dichroism melting studies. Overall this manuscript presents results on the molecular interaction from structural and energetic perspectives providing an in depth insight into drug-protein interaction.

Spectroscopic and calorimetric investigations on the binding of phenazinium dyes safranine-O and phenosafranine to double stranded RNA polynucleotides.

RNA targeting through small molecules that can selectively bind specific RNA structures is an important current strategy in therapeutic drug development. Towards this strategy a comparative study on the interaction of two phenazinium dyes, safranine-O and phenosafranine to double stranded RNAs, poly(I).poly(C), poly(A).poly(U) and poly(C).poly(G) was performed. Spectrophotometric and spectrofluorimetric studies revealed non-cooperative binding of the dyes to the duplex RNA with binding constants of the order 10(5)M(-1) with a higher affinity of safranine-O to poly(I).poly(C) followed by poly(A).poly(U) and poly(C).poly(G). Anisotropy and fluorescence quenching results confirmed an intercalation mode of binding for the dyes on these RNAs. Binding induced conformational changes in the RNA polynucleotides were revealed from circular dichroism data. Thermal melting study and DSC experiments demonstrated stabilization of dye-RNA complexes. Calorimetric studies revealed that the binding was accompanied by a large positive entropy term with a small negative enthalpy contributions. Significant hydrophobic forces in the complexation of the double stranded RNAs with the dyes were confirmed from the negative heat capacity changes. Enthalpy-entropy compensation was also observed in the binding. Parsing of the Gibbs energy suggested a larger non-electrostatic contribution in all the cases. The results presented here may be helpful to design new types of RNA-based therapeutic agents.

Binding of fluorescent acridine dyes acridine orange and 9-aminoacridine to hemoglobin: Elucidation of their molecular recognition by spectroscopy, calorimetry and molecular modeling techniques.

The molecular interaction between hemoglobin (HHb), the major human heme protein, and the acridine dyes acridine orange (AO) and 9-aminoacridine (9AA) was studied by various spectroscopic, calorimetric and molecular modeling techniques. The dyes formed stable ground state complex with HHb as revealed from spectroscopic data. Temperature dependent fluorescence data showed the strength of the dye-protein complexation to be inversely proportional to temperature and the fluorescence quenching was static in nature. The binding-induced conformational change in the protein was investigated using circular dichroism, synchronous fluorescence, 3D fluorescence and FTIR spectroscopy results. Circular dichroism data also quantified the α-helicity change in hemoglobin due to the binding of acridine dyes. Calorimetric studies revealed the binding to be endothermic in nature for both AO and 9AA, though the latter had higher affinity, and this was also observed from spectroscopic data. The binding of both dyes was entropy driven. pH dependent fluorescence studies revealed the existence of electrostatic interaction between the protein and dye molecules. Molecular modeling studies specified the binding site and the non-covalent interactions involved in the association. Overall, the results revealed that a small change in the acridine chromophore leads to remarkable alteration in the structural and thermodynamic aspects of binding to HHb.